Recombinant expression systems will be established for papain family cysteine proteases that have been implicated in parasitic diseases. In particular, cysteine proteases of protozoan and helminth parasites including Trypanosoma cruzi, Trypanosoma. brucei, Leishmania donovani and Plasmodium falciparum will be expressed in E. coli, P. pastoris, and/or mammalian cell expression systems. The extended substrate specificity of these enzymes will be determined using combinatorial methods that can identify the preferred peptide substrates for the binding pockets both N-terminal and C-terminal to the scissile peptide bond. Optimal substrates will be identified for the proteases and used to assist in high throughput screening efforts of the program project to identify inhibitors. Furthermore, the specificity profiling information will be combined with data from related cysteine proteases to identify key amino acids that line the binding pocket and serve as putative determinants of substrate specificity. Site directed substitutions will be made and the variant enzymes profiled to test the role of these amino acids in substrate recognition and enzyme turnover. Using cruzain as a strategic template for the other cysteine proteases, peptide-based acyloxymethyl ketone inhibitors and single chain antibody inhibitors will be made that are highly specific and map the extended substrate binding pockets. These inhibitors will be used to probe the active site of the enzyme in solution using NMR spectroscopy. Cruzain will be uniformly labeled and/or labeled at specific amino acids with 13C/15N. NMR will be used to determine if conformational flexibility exists in the active site and binding pockets of cruzain when free in solution and upon binding different inhibitors. The active site histidine will also be monitored in NMR experiments that measure the coupling constants of the imadazole ring to follow the protonation state and tautomeric state of the histidine. Correlating these changes with inhibitor binding will assist in the concerted efforts of the program project to develop cysteine protease inhibitors as new anti-parasitic drugs.